PROJECT SUMMARY Memory CD8+ T cells are essential cells of the immune system, retained post infection to protect from subsequent immunological insults. Many transcriptional regulators and some proximal signaling cascades associated with both naive and memory T cells have been described and have greatly informed the process by which immunological memory is formed. Despite clear differences in transcription factor expression and cytokine requirements, how global signal transduction induced by antigen binding differs between naive and memory CD8+ T cells is completely unknown and thus represents a significant gap in our knowledge base. One key signaling event that occurs in response to TCR stimulation is the activation of kinases that phosphorylate tyrosine residues on downstream signaling proteins. Therefore, comparing the phosphotyrosine signaling networks between naive and memory T cells will identify targets for modulation of the CD8+ T cell immune response to positively influence response to disease and vaccine efficacy. The objective of this specific proposal is to determine the intracellular signaling pathways that are unique to restimulated memory CD8+ T cells. Our central hypothesis is that distinct signal transduction pathways regulate the memory CD8+ T cell response. The rationale and scientific premise for the proposed work is that understanding at a global proteomic level the changes that occur in T cells as they differentiate from naive to memory T cells will enable more efficacious vaccine and drug treatment design to combat disease and infection. We will test our central hypothesis with the following specific aims: 1) Determine the TCR signal transduction changes that occur in CD8+ nave and memory T cells. To profile kinase signaling networks, we will utilize a highly sensitive quantitative mass spectrometric assay we developed on naive, effector and memory CD8+ T cells. Based on a preliminary screen, we identified that Jak2 is hyperphosphorylated, indicating it is a key mediator of the recall response. We will test the hypothesis that Jak2 hyperactivation is critical for memory recall responses. Together, the completion of Aim1 will identify novel signaling networks that contribute to CD8+ memory recall responses and 2) Defining phosphorylation of CREB binding protein (CBP) as a molecular switch that regulates transcriptional networks in memory CD8+ T cells. Given our preliminary data in which we uncovered unique signaling pathways associated with CD8+ memory T cell formation, we hypothesize that in memory CD8+ T cells, increased Jak2 activation drives phosphorylation of CBP to promote a unique transcriptional landscape versus a nave T cell. We will identify how phosphorylation of CBP regulates binding of transcription factors such as CREB and MYB. Additionally, we will take a genetic approach to determine how CBP functions in the formation of CD8+ memory cells and CD8+ memory recall responses. Together, data obtained in this Aim2 will establish a novel mechanism that explains how changes in tyrosine kinase activity associated with CD8+ T cell memory formation drive unique transcriptional programs.